When particulates contained in fluid are placed in a centrifugal field, each particulate undergoes a force corresponding to its mass. When this force is larger than the force of self-diffusion, the particulates are separated from the liquid layer to form a solid layer. When the particulates are small, however, they do not settle fully but form a layer at a location where the centrifugal force is balanced by the force of self-diffusion. When fluid flows through a narrow channel, the flow rate of the fluid at the portion where the fluid is in contact with the inner wall of the channel is lower than the flow rate of the central portion. Hence, the distribution of flow assumes a parabolic curve whose vertex is located at the center of the channel. When a centrifugal field is applied perpendicularly to the channel, the particulates in the fluid move at flow rates that are inherent in the positions at which the centrifugal force is balanced by the force of self-diffusion. The technique employing this principle to separate particulates in fluid is known as sedimentation field-flow fractionation (SFFF). Columns used for this fractionation must satisfy the following requirements:
(1) The wall must be made from a chemically inactive material, and must have a high degree of flatness. The roughness of the wall surface must have submicron dimension.
(2) A channel that is used for centrifugal separation must have a width and a height which are accurately uniform in the direction of flow of fluid.
(3) The inlet port of the column must be fully isolated from the outlet port.
(4) There must be no leakage.
(5) The dead volume of the inlet and outlet ports must be minimized.
(6) The column must be easily disassembled and reassembled for cleaning of the inside.
Kirkland and others have made some proposals to meet these requirements.
FIGS. 1(a)-1(c) show a conventional column for a continuous particle fractionation apparatus making use of a centrifugal field. As shown in FIG. 1(a), this column comprises an inner ring 21 and an outer ring 22. The inner ring 21 is provided with an inlet port 23 and an outlet port 24, and is provided with a machined groove 25 at a position located between these ports. The outer surface of the inner ring 21 has a separation groove 25 extending from end to end. Two seal grooves 27 are formed on opposite sides of the separation groove 25. When the column is assembled, a seal material 28 is inserted in the seal grooves 27. Then, the inner ring 21 is inserted into the outer ring 22, as shown in the cross section of FIG. 1(b). Thus, a channel 29 is formed in the separation groove 25 which is surrounded by the inner ring 21 and the outer ring 22. A wedge 30 is used to expand the inner ring 21 to couple it to the outer ring 22.
The channel 29 measures 50 to 300 .mu.m in width, 25 mm in height, and 58 cm in length, for example. The dimensions of the cross section of the channel 29 are uniform over its whole length. The inlet port 23 for injecting fluid into the channel 29 and the outlet port 24 for taking the fluid out of the channel 29 are disposed very close to each other at both ends of the channel 29. The arrangement of the inlet port 23, the outlet port 24, the separation groove 25, and the seal grooves 27 is shown in FIG. 1(c).
To effect sedimentation field-flow fractionation, fluid is passed through the channel 29 of the column as shown in FIGS. 1(a)-1(c) at a flow rate of 1 ml/min. The total capacity of the channel 29 is about 3 ml. If the liquid leaks from the column, forming a bypass passage, then the peak appearing on an obtained graph will deviate from its correct location or will not indicate a correct value. The sample which moves at a rate slower by two orders of magnitude than the average flow rate of the fluid flowing through the column is spaced a distance of the order of microns from the wall surface. Accordingly, if the wall surface has roughness of the order of microns, then the analytical accuracy is greatly affected. For these reasons, it is important for the column used for sedimentation field-flow fractionation to prevent leakage of liquid, to secure a seal pressure and to secure high accuracy in machining the surface of the channel 29.
However, the conventional column requires highly sophisticated machining techniques to machine the channel surface accurately and to prevent leakage of liquid, because the separation groove constituting the channel as shown in FIGS. 1(a)-1(c) is formed in the outer surface of the inner ring. Another problem is that it is difficult to assemble the column, because the difference between the outside diameter of the inner ring and the inside diameter of the outer ring is quite small.